Spironolactone has produced beneficial effects in animal models of neurodegenerative disorders. However, the underlying mechanisms of this agent on neurons and glia are mostly unknown. Therefore, we aimed to show the effects of spironolactone and fludrocortisone, a mineralocorticosteroid receptor agonist, on neuronal and glial toxicity induced by N-methyl-D-aspartate (NMDA) activation and chloroquine, an autophagy inhibitor, in the cell culture. We exposed the SHSY5Y neuroblastoma and 1321N1 astrocytoma exposed to NMDA (25µM), or chloroquine (40µM) for 24 and 48h to induce neuronal and glial toxicity. Spironolactone (1, 10, and 20µM) or fludrocortisone (300nM) were also added to the cells for 24 and 48h. Cell survival was measured using the MTT assay. Neurons and astrocytes treated with NMDA and spironolactone (1, 10, and 20µM) for 24 and 48h had lower cell death compared with the NMDA-treated group. Moreover, cells treated with NMDA and fludrocortisone for 24 and 48h had higher viability in comparison to the NMDA-treated group. The neuronal cells treated with chloroquine and spironolactone (10 and 20µM) for 24h had higher cell viability compared with the chloroquine group. Chloroquine plus spironolactone (20µM) treatment for 24 and 48h increased cell viability of astrocytes compared with the chloroquine-treated group. Moreover, the treatment of neurons and astrocytes with chloroquine plus fludrocortisone for 24h decreased cell death.  Spironolactone and fludrocortisone protected neurons and astrocytes against NMDA- and chloroquine-induced toxicity. The mechanism of neuronal and glial protective effects of spironolactone possibly related to the inhibition of the mineralocorticosteroids. However, spironolactone might affect other non-mineralocorticoid systems.